The present invention relates to methods for coupling ligands such as bindable biological substances to polymeric materials. The resulting ligand-polymer conjugates are particularly useful as reagents in binding assays, e.g., immunoassays. More particularly, the invention concerns such coupling methods wherein steps are taken to control in a reproducible manner the number or density of ligands that become linked to the polymeric material.
There is a continuing need for improved methods of chemically coupling ligands to polymeric supports, particularly in the preparation of immunoassay reagents. Analytical performance is often dependent upon how well characterized and reproducible such reagents can be prepared. This is particularly true for the agglutinator reagent in an immunoturbidimetric assay.
The typical protocol for performing an immunoturbidimetric assay involves setting up a competitive binding reaction between the analyte of interest in the test sample and the agglutinator reagent for binding to antibody against the analyte. The agglutinator reagent comprises a polymer support bearing multiple ligands capable of binding with a particulate reagent bearing anti-analyte antibody. Binding between multiple agglutinator reagent molecules and multiple anti-analyte particles produces an agglutination that is detectable by turbidimetry. Since analyte in the sample competes with this agglutination reaction, as analyte concentration increases, the turbidity of the reaction mixture decreases.
Assay performance of the immunoturbidimetric system is dependent upon the consistency of the agglutinator reagent. If the number of ligand moieties coupled to individual polymers varies widely in the reagent population, the size and rate of formation of light scattering centers produced upon agglutination will similarly vary widely. Such variance can introduce a significant error factor in the precision of the assay. Likewise assay sensitivity is affected by variability in the agglutinator reagent. If the average number of ligands per polymer is low, the maximum agglutination in the absence of analyte is of limited detectability. On the other hand, if the ligand density is too high, it becomes difficult for low levels of analyte to effectively compete for binding to the antibody reagent.
The prior art attempts to control the number or density of ligands coupled to polymer supports have depended essentially on the ability to control all reaction conditions affecting the coupling reaction such as reactant concentrations and purity, temperature, pH, and time of reaction. The ability to control so many different aspects of the coupling process is not practical considering the types of polymeric materials that are conventionally used. Natural biopolymers such as bovine serum albumin as well as synthetic materials have been used for the preparation of multivalent ligand-polymer conjugates. However, such polymeric materials generally have limited available coupling sites as well as reactivity that is sensitive to slight changes in coupling conditions.